Behavioural Brain Research 305 (2016) 100–107 Contents lists available at ScienceDirect Behavioural Brain Research jou rnal homepage: www.elsevier.com/locate/bbr Research report Conessine, an H3 receptor antagonist, alters behavioral and neurochemical effects of ethanol in mice a,b,c c a,b,∗ Gessynger Morais-Silva , Mariane Ferreira-Santos , Marcelo T. Marin a Laboratory of Pharmacology, School of Pharmaceutical Sciences, Univ Estadual Paulista—UNESP, Araraquara, SP, Brazil b Joint Graduate Program in Physiological Sciences, UFSCar/UNESP, São Carlos/Araraquara, SP, Brazil c Institute of Biomedical Sciences, Federal University of Uberlândia (UFU), Uberlândia, MG, Brazil h i g h l i g h t s • Conessine, an H3 receptor antagonist, exacerbated ethanol-induced psychostimulation. • Pretreatment with conessine did not alter ethanol CPP. • Conessine had reinforcing proprieties per se. • Norepinephrine and serotonin might be related to the reinforcing proprieties. • Dopamine might be related to the conessine exacerbation of ethanol psychostimulation. a r t i c l e i n f o a b s t r a c t Article history: Ethanol abuse potential is mainly due to its reinforcing properties, crucial in the transition from the Received 23 November 2015 recreational to pathological use. These properties are mediated by mesocorticolimbic and nigrostriatal Received in revised form 9 January 2016 dopaminergic pathways and neuroadaptations in these pathways seem to be responsible for addiction. Accepted 22 February 2016 Both pathways are modulated by other neurotransmitters systems, including neuronal histaminergic sys- Available online 26 February 2016 tem. Among the histamine receptors, H3 receptor stands out due to its role in modulation of histamine and other neurotransmitters release. Thus, histaminergic system, through H3 receptors, may have an Keywords: important role in ethanol addiction development. Aiming to understand these interactions, conessine, an Alcohol addiction Psychostimulation H3 receptor antagonist, was given to mice subjected to the evaluation of ethanol-induced psychostim- ulation, ethanol CPP and quantification of norepinephrine, dopamine, serotonin and their metabolites Conditioned place preference Monoamines in mesocorticolimbic and nigrostriatal pathways following acute ethanol treatment. Systemic cones- H3 receptor antagonist sine administration exacerbated ethanol effects on locomotor activity. Despite of conessine reinforcing Conessine effect on CPP, this drug did not alter acquisition of ethanol CPP. Ethanol treatment affects the sero- toninergic neurotransmission in the ventral tegmental area, the dopaminergic neurotransmission in the pre-frontal cortex (PFC) and caudate-putamen nucleus (CPu) and the noradrenergic neurotransmission in the CPu. In the PFC, conessine blocked ethanol effects on dopaminergic and noradrenergic neurotrans- mission. The blockade of H3 receptors and ethanol seem to interact in the modulation of dopaminergic neurotransmission of nigrostriatal pathway, decreasing dopamine metabolites in substantia nigra. In con- clusion, conessine was able to change psychostimulant effect of ethanol, without altering its reinforcing properties. This exacerbation of ethanol-induced psychostimulation would be related to alterations in dopaminergic neurotransmission in the nigrostriatal pathway. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Every year the abuse of alcoholic beverages is responsible for about 3.3 million deaths [1]. Several injuries are causally linked to ∗ Corresponding author. Current address: Depto. Princípios Ativos Naturais e Toxi- alcohol use and ethanol addiction is the major risk factor for the cologia (PANT), Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista most of them [2]. The abuse potential of psychoactive substances, (UNESP), Rod. Araraquara-Jaú-km 01, CEP 14800-903 Araraquara, SP, Brazil. including ethanol, is mainly due to their reinforcing proprieties, E-mail addresses: [email protected], [email protected] a characteristic responsible for the initial search and repeated (M.T. Marin). http://dx.doi.org/10.1016/j.bbr.2016.02.025 0166-4328/© 2016 Elsevier B.V. All rights reserved. G. Morais-Silva et al. / Behavioural Brain Research 305 (2016) 100–107 101 consumption of these substances that can lead to alcohol-related Uberlândia, MG-BRA; 30–35 g) were transferred to our animal facil- injuries and development of addiction in susceptible subjects. ity at least seven days before the start of the experiments and The most important neural pathway related to the reinforcing were housed within groups of four or five per cage. The room was ◦ proprieties, whether for natural stimuli or drugs of abuse is the maintained at a temperature of 23 ± 2 C on a 12:12 h light/dark dopaminergic mesocorticolimbic pathway. It consists of dopamin- cycle with ad libitum water and food access. All experiments were ergic neurons located in ventral tegmental area (VTA) that project performed during the light phase of the cycle and animals were to the nucleus accumbens (NAc) and pre-frontal cortex (PFC) [3]. randomly tested across this time period. The experimental protocol Other dopaminergic pathway important in the processes related to was approved by the Ethical Committee for the Animal Utilization addiction is the nigrostriatal pathway, which is comprises neurons of Federal University of Uberlândia (CEUA 029/13) and the experi- from the substantia nigra (SN) pars compacta to the caudate- ments were conducted according to the principles of the National putamen nucleus (CPu). This last neuronal pathway and theirs Council for Animal Experiments Control (CONCEA), based on NIH connections have an important role in habit formation, crucial fea- Guidelines for the Care and Use of Laboratory Animals. ture of addiction development [4,5]. Among the aminergic systems that exist in mammalian brain, 2.2. Drugs the histaminergic system acts modulating sensory information according to individual’s memories and physiology. It consists of Conessine (Sigma Aldrich, St. Louis, MO-USA) was diluted in a small neuronal group that projects to the entire nervous system. extra virgin olive oil (Sandéleh Alimentos, Sorocaba, SP-BRA) as The histamine containing neurons are restricted to the posterior vehicle and administered subcutaneously (0.1 mL/10 g) at the doses part of the hypothalamus, the tuberomammilary nucleus, which of 0.1, 1.0 or 10.0 mg/kg. Ethanol was diluted in saline (NaCl 0.9%) sends projections to both mesocorticolimbic and nigrostriatal path- and administered intraperitoneally at the dose of 2.0 g/kg in the ways. Four receptors are known for histamine, named H1–4. They evaluation of the locomotor activity and neurotransmitters quan- are G-protein coupled receptors highly distributed in central ner- tification experiments and at the dose of 1.0 g/kg in conditioned vous system [6]. The H3 receptor acts mainly as an inhibitory place preference (CPP) experiment. The drug injected dose was autorreceptor in the central nervous system through Gi/0 protein. It obtained from 20% (v/v) ethanol solution in the experiments for controls the release of many neurotransmitters in addition to his- the evaluation of locomotor activity and from 10% (v/v) ethanol tamine, such as dopamine, norepinephrine, serotonin, GABA and solution in the CPP experiment. acetylcholine. This receptor is highly expressed in addiction-related brain areas, such as NAc and PFC [6]. There are many H receptor antagonists available for research 3 2.3. Evaluation of locomotor activity use. Among them, conessine is one of the most selective for H3 receptors, with high affinity. It’s derived from the bark and seeds of Locomotor activity was evaluated by the measurement of the plants from Apocynaceae family, especially Holarrhena antidysen- distance travelled (in meters) by mice after treatments in the open terica, freely crosses blood-brain barrier and has slow clearence field (OF) apparatus. The OF is a circular arena with black floor (Mas- in central nervous system [7,8]. Different from the most common ter One, Ribeirão Preto-SP, Brazil), 30 cm in diameter, surrounded compounds, conessine is a non-imidazole H antagonist, proba- 3 by 30 cm high transparent walls. bly having no action in enzymes of cytochrome P450 [9]. This is In the day of the experiment, the animals received conessine a desirable characteristic of this drug for the study of mechanisms (0.1, 1.0 or 10.0 mg/kg) or vehicle and one hour later ethanol involved in ethanol addiction, since it probably does not affect alco- (2.0 g/kg) or saline. The animals (N = 8–9 animals per group) were hol metabolism. then immediately placed in the OF and their locomotor activity Post-mortem studies using the brain of alcoholics suggest a role measured during 30 min by the behavioral analysis software ANY- of the histaminergic system on ethanol addiction. The brain of such maze (Stoelting Co., Wood Dale, IL-USA). Animal’s movements were individuals has increased histamine metabolites [10]. Furthermore, captured by a HD camera fixed above four OF apparatuses and some polymorphisms were found in genes that codify enzymes of connected to a computed with the behavioral analysis software. histamine synthesis and metabolism in ethanol addicted [11,12]. In Distance travelled was analyzed as total distance covered during animal models of substance abuse, drugs which act modulating his- the 30 min and also in blocks of 5 min for a temporal analysis of taminergic system both aggravate and ameliorate